ATIII is a major inhibitor of enzymes in the coagulation cascade, including thrombin (Rosenberg and Damus, (1973) J. Biol. Chem., 248, 6490-6505) and factor Xa (Kurachi et al., (1976) Biochemistry, 15, 373-377). Many hereditary mutations in ATIII have been identified that promote hypercoagulability because of unchecked activity of the coagulation enzymes (Reviewed in van Boven and Lane, (1997) Semin. Hematol., 34, 188-204). Acquired deficiencies of ATIII can also occur with negative repercussions on hemostasis, as for example during septic disseminated intravascular coagulopathy (DIC) (Bick et al., (1980) Am. J. Clin. Path., 73, 577-583); (Buller and Cate, (1989) Am. J. Med., 87, 44S-48S); (Damus and Wallace, (1989) Thromb. Res., 6, 27); (Hellgren et al., (1984) Intensive Care Med., 10, 23-28); (Lammle et al., (1984) Am J Clin Pathol, 82, 396-404); (Mammen et al., (1985) Semin. Thromb. Hemost., 11, 373-383). In contrast, hemorrhage resulting from excess inhibition of blood coagulation by ATIII can occur in the presence of pharmaceutical heparin, which is frequently used to treat and prevent hypercoagulable states Giant et al., (1977) Lancet, 1, 1133-1135).
ATIII is a 432 amino acid Mr 58 000 plasma glycoprotein (Bock et al., (1982) Nucleic Acids Res., 10, 8113-8125); (Petersen et al., (1979) The Physiological Inhibitors of Coagulation and Fibrinolysis (pp. 43-54): Elsevier/North Holland Biomedical Press) which not only inhibits thrombin and factor Xa, but also serine proteinases preceding them in the intrinsic pathway (e.g., factor IXa, factor XIa, factor XIIa) (Rosenberg, (1977) Fed. Proc., 36, 10-18) and the extrinsic pathway (factor VIIa-TF) (Lawson et al., (1993) J. Biol. Chem., 268: 767-770); (Rao et al., (1993) Blood, 81: 2600-2607) of blood coagulation. Factor VIIa-TF has roles not only in coagulation and thrombosis, but is implicated in cancer angiogenesis and metastasis as well (Ruf and Mueller, (1996) Curr. Opin. Hematol., 3: 379-84). ATIII also effects non-coagulant, thrombin-mediated pathways, such as thrombin-induced smooth muscle cell proliferation (Hedin et al., (1994) Arterioscler. Thromb., 14: 254-260) and thrombin-mediated neutrophil extravasation (Ostrovsky et al., (1997) Circulation, 96: 2302-2310). Moreover, ATIII promotes endothelial release of prostacyclin (PGI2), which inhibits leukocyte and platelet activation, and has vasodialator properties (Uchiba et al., (1997) Seminars in Thrombosis and Hemostasis, 23: 583-590).
The inhibitory activity of ATIII towards its target enzymes is dramatically enhanced by heparin (Rosenberg and Damus, (1973) J. Biol. Chem., 248, 6490-6505) and vascular surface heparan sulfate proteoglycans (HSPGs) (Marcum et al., (1983) Am. J. Physiol., 245: H725-733) The, heparin binding property of antithrombin directs ATIII to sites where its target enzymes are generated, and potentiates its activity on these surfaces.
Antithrombin is synthesized in the liver and secreted in the blood as two different isoforms (Peterson and Blackburn, (1985) J. Biol. Chem., 260, 610-615). The predominant species (90%), α-ATIII, has four identical N-glycosidic-linked polysaccharide chains attached to asparagine residues 96, 135, 155, and 192 (Franzen et at., (1980) J. Biol. Chem., 255, 5090-5093); (Mizuochi et al., (1980) Arch. Biochem. Biophys., 203, 458-465). The minor β-ATIII isoform (10%) lacks the oligosaccharide side chain at asparagine 135 (Brennan et al., (1987) FEBS Lett., 219, 431-436). The β-glycoform lacks a carbohydrate on Asn-135 because of inefficient glycosylation of the NXS consensus sequence (Picard et al., (1995) Biochemistry, 34, 8433-8440). U.S. Pat. Nos. 5,618,713 and 5,7000,663 disclose that mutation at one or more glycosylation sites (for example Asn 135, Asn 155) increases the heparin-binding/heparin-activating properties while retaining the protease specificity of ATM. In particular, those patents disclose and claim modified ATIIIs with replacement of asparagines in N-glycosylation sites by residues which are incapable of being glycosylated. U.S. Pat. Nos. 5,618,713 and 5,700,663 do not disclose the present ATIIIs with improved resistance to human neutrophil elastase or enhanced heparin affinity due to mutation of the third position in N-glycosylation sequences.
Human neutrophil elastase cleaves and inactivates ATIII (Jochum et al., (1981). Hoppe-Seyler's Z. Physiol. Chem., 362, 103-112). The reported neutrophil elastase cleavage sites were after the P5-Val and P4-Ile (Carrell and Owen, (1985) Nature, 317, 730-732). Furthermore, Jordan and colleagues showed that elastase inactivation of ATIII was heparin dependent (Jordan et al., (1987) Science, 237, 777-779). It has been hypothesized that elevated elastase (Nuijens et al., (1992) J. Lab. Clin. Med., 119, 159-168) is responsible for the inactivation of ATIII in sepsis (Seitz et al., (1987) Eur. J. Haematol., 38, 231-240) and reduced antithrombin levels in septic DIC (Bick et al., (1980) Am. J. Clin. Path., 73, 577-583); (Buller and ten Cate, (1989) Am. J. Med., 87, 44S-48S); (Damus and Wallace, (1989) Thromb. Res., 6, 27); (Hellgren et al., (1984a) Intensive Care Med., 10, 23-28); (Lammle et al., (1984) Am J Clin Pathol, 82, 396-404); (Mammen et al., (1985) Semin. Thromb. Hemost., 11, 373-383). This acquired decrease in functional ATIII would contribute to the progression of DIC due to the inability to inhibit activated coagulation proteinases, ultimately leading to thrombin activation, fibrin formation and coagulation factor consumption.
Several animal and human studies have suggested that ATIII concentrate therapy may be effective in reducing mortality rates of patients suffering from septic disseminated intravascular coagulopathy (DIC). Using an endotoxemic rat model, (Emerson et al. (1987) Am. J. Med., 87, 27S-33S) have shown that prophylactic ATIII treatment affords protection from the decline of hemostasis associated with septicemia complicated by DIC. ATIII treatment has also been found to be effective in reducing mortality and stabilizing hemostatic parameters when administered after the presence of DIC has been established in Klebsiella pneumoniae—induced septicemic rats (Dickneite and Paques, (1993) Thromb. Haemost., 69, 98-102). Human studies of ATIII replacement therapy have also shown promising results. Patients with septic shock and DIC showed improved survival as well as improved hematologic characteristics and organ function parameters with ATIII substitution (Blauhut et al., (1985) Thromb. Res., 39, 81-89); (Delshammar et al., (1989). J. Intern. Med., 225, 21-27); (Fourrier et al., (1993) Chest, 104, 882-888); (Hellgren et al., (1984b) Thromb. Res., 35, 459-466); (Jochum, (1995) Semin. Hematol., 32, 19-32). Review of the various patient trials showed a survival rate ranging from 64-97% (combined, 76%) among those receiving ATIII replacement, compared to a survival range of 7.6-25% (combined, 19%) (Vinazzer, (1995) Clin. Appl. Thrombosis/Hemostasis, 1,62-65). These studies showed promising responses to ATIII concentrates in the treatment of septic DIC. However, very large doses of ATIII were required (90-120 U/kg/day) (Fourrier et al., (1993) Chest. 104, 882-888); (Jochum, (1995) Semin. Hematol., 32, 19-32). This finding was consistent with continued inactivation of the exogenous ATIII by elevated levels of neutrophil elastase. These observations suggested that reversal of septic DIC may be achievable using lower doses of recombinant ATIII variants with engineered resistance to the neutrophil proteinases elastase, cathepsin G and proteinase-3.
Previous attempts at replacing the elastase cleavage site with non-cleavable residues has resulted in impaired thrombin binding inhibition. The authors “report that the reiteration of the substitution best fitting these criteria, that of Trp at both P4 and P5 [residues 389 and 390], does not confer significant LE [neutrophil elastase] resistance on AT.” Cunningham et al, 88 Thrombsis Res 171 (1997). These modified ATIIIs were considered commercially available compared to wild-type ATIII.
WO 91/00291 also discloses modified antithrombin III variants. It broadly describes modified ATIIIs wherein “at least one amino acid from the region comprising amino acids 384-396 is replaced by the corresponding amino acids group around the factor Xa cleavage site in factor II related to the formation of meizothrombin . . . ” The present ATIIIs were not mentioned in that publication.
Citation of the above documents is not intended as an admission that any of the foregoing is prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of the documents.